The long-term objectives of this project are (1) to identify the specific regions within voltage-gated sodium channels that govern channel activation, and (2) to develop a unified model of local anesthetic interaction with sodium channels that includes channel activation and inactivation parameters. Given the importance of channel gating to receptor modulation and local anesthetic block, a better understanding of the channel gating mechanism will be a critical step toward explaining why clinically important local anesthetics, such as bupivacaine and lidocaine, affect cardiac physiology at circulating concentrations that have little effect on skeletal muscle physiology. Numerous studies have used site-directed mutagenesis techniques to alter the kinetic phenotypes of sodium channel isoforms cloned from cardiac and skeletal muscle, yet no study has identified the channel regions responsible for the more hyperpolarized activation and inactivation ranges of cardiac sodium channels as compared with skeletal muscle sodium channels. To date, careful screening of the amino acid sequences of cardiac and skeletal muscle sodium channels and "residue swapping" between the two isoforms at suspected regions of importance to channel gating have had limited success. This proposal uses an alternative approach to address questions of isoform-specific gating in sodium channels. Neurotoxins that bind to sites 2, 3, and 4 on voltage gated sodium channels alter channel kinetics and other channel properties such as ionic selectivity. Whole-cell sodium currents through human heart (hH1) and rat skeletal (mu1) muscle sodium channels will be compared before and after modification by neurotoxins. The goal is to identify isoform-specific changes in channel kinetics, ionic selectivity, and local anesthetic interactions at toxin-modified channels. Because several residues critical for neurotoxin binding have already been localized, isoform-specific alterations in channel behavior after toxin modification can be used to target channel regions that are potentially important to channel gating. Analyses of the differences between toxin-modified hH1 and u1 channels will be used to design mutant channels and channel chimeras to further narrow the search for channel regions that are important in channel gating. Once the gating mechanism of the channels has been deduced, current models of local anesthetic block can be modified to explain the elevated sensitivity of cardiac tissue to antiarrhythmic and cardiotoxic local anesthetics.